Research in the Immunopathology Section focuses on the biological mediators and signal transduction pathways involved in the modulation of human monocyte and lymphocyte functions that may contribute to the immunopathology associated with various inflammatory lesions. Monocytes/macrophages are prominent in many inflammatory diseases, such as periodontal disease, rheumatiod arthritis, atherosclerosis, and cancer. The pathology associated with these diseases involves alterations in the integrity of the connective tissue framework implicating a role for matrix metalloproteinases (MMPs). MMPs are comprised of a family of zinc dependent endopeptidases divided into major subgroups that include the interstitial collagenases, the gelatinases, stromelysins, membrane type MMPs and others. Collectively these enzymes are capable of degrading all the extracellular matrix components. Because MMPs and tissue inhibitors of MMPs (TIMPs) are believed to play a major role in the destruction and remodeling of connective tissue, a major emphasis has been placed on how these enzymes and inhibitors are regulated in the human monocyte. [unreadable] [unreadable] The objectives of the research in the Immunopathology Section are to: (1) study the relationship between biological mediators, such as cytokines and biomarkers, commonly found at disease sites and their impact on MMP and cytokine production by monocytes; (2) determine the early and late components in the signal transduction pathway leading to the activation of monocytes and the production of MMPs; (3) investigate the potential role of the monocyte in the activation of MMPs produced by other cell types; (4) determine mechanisms that regulate the production of immunomodulatory molecules by monocytes; (5) investigate how products produced by monocytes regulate cellular functions of immune cells and cancer cells, and (6) where appropriate use animal models and/or clinical studies to determine the potential role of the monocyte in disease by examining: a) whether the administration of potential disease modifying agents affect monocyte function and b) the relationship of risk factors and polymorphisms in disease and monocyte function.[unreadable] [unreadable] We have recently published our findings on the role of the urokinase-type plasminogen activator (uPA)/plasminogen system in the induction of MMP-1 synthesis by monocytes. The data from this study demonstrate that catalytically active high MW uPA, which binds to the uPA receptor (uPAR), and low MW uPA, which does not, significantly enhanced MMP-1 synthesis by activated human monocytes. In contrast, the amino- terminal fragment of uPA, which binds to uPAR but lacks the catalytic site, failed to induce MMP-1 production indicating that uPA-stimulated MMP-1 synthesis was plasmin-dependent. Endogenous plasmin generated by the action of uPA or exogenous plasmin increased MMP-1 synthesis by signaling through annexin A2 as demonstrated by inhibition of MMP-1 production with antibodies against annexin A2 and S100A10, a dimeric protein associated with annexin A2. Interaction of plasmin with annexin A2 resulted in the stimulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen-activated kinase p38 (p38 MAPK), cyclooxygenase-2 and PGE2 leading to increased MMP-1 production. Furthermore, binding of inactive plasmin to annexin A2 inhibited plasmin-induction of MMP-1 suggesting the inactive plasmin may be useful in suppressing inflammation. Current studies stemming from these findings include: plasminogen expression in monocytes and its role in activation of pro-MMP-1; the effect of soluble uPAR (suPAR) released from monocytes as a feed back mechanism on signaling through the G protein-coupled chemotactic receptor, formyl-methyonyl-leucyl-proline-like receptor-1 (FPRL-1): and the potential role of plasmin and inactive plasmin in the regulation of MMP production by tumor cells, the interaction between monocytes and tumor cells, tumor progression and wound healing in a murine model systems.[unreadable] [unreadable] Unlike other cell types in which secreted MMP-1 is only detected in the proform, MMP-1 in supernatants of stimulated monocytes is in the active form. PCR analysis has shown that monocytes express plasminogen which accounts for the generation of endogenous plasmin leading to the activation of proMMP-1. These findings demonstrate that the plasmin secreted by monocytes as they enter an inflammation site may play an important role in the activation of proMMP-1 produced by resident macrophages and other cell types, such as fibroblasts and tumor cells. However, as monocytes mature into macrophages MMP-1 is detected mainly in the proform. We are currently analyzing whether gene expression of plasminogen is lost and/or inhibitors of plasmingogen activation are increased during the maturation of monocytes to macrophages. [unreadable] [unreadable] One of the actions of plasmin and other proteases is the cleavage of uPAR resulting in a soluble D2D3 fragment. In collaboration with Dr. Michael Plow at the Finsen Laboratory in Copenhagen we are examining whether D2D3 through its interaction with FPRL-1 may regulate MMP production through a feedback mechanism. This study is being conducted with antibodies against FPRL-1, FPRL-1 siRNA and agonist or antagonists of FPRL-1. The present findings indicate that FPRL-1 engagement may serve as an amplification loop for MMP-1 production.[unreadable] [unreadable] Monocyte/macrophage interaction with tumor cells and the role of plasmin in this process is believed to be an important determinant in tumor progression. To address this we are currently studying the interaction between primary monocytes and macrophage subtypes with head and neck squamous cell carcinoma cells lines in the production of MMPs and the role of plasmin and inactive plasmin in this process. [unreadable] [unreadable] Identification of cancer fighting agents in food products has been an ongoing interest in research. Cranberry fruit and juice have been reported to have multiple health benefits including anticancer effects. In collaboration with Scientists from Israel we examined the effect of a nondialyzable material (NDM) fraction with a molecular weight of 12-30K derived from cranberry juice on the proliferation and invasion through Matrigel of murine Rev-2-T-6 lymphoma cells. NDM (12-30K) inhibited the proliferation and invasion of the lymphoma cells. Subsequent experiments carried out in Israel showed that this fraction inhibited the growth of Rev-2-T-6 tumors in vivo and enhanced the generation of antilymphoma antibodies. Present studies are focused on characterizing this fraction for potential use as an anticancer agent and ability to regulate MMP production by monocytes.[unreadable] [unreadable] Monocytes/macrophages respond to the lipopolysaccharide (LPS) of bacteria through specific recognition receptors triggering inflammatory responses. Toll-like receptor 4 (TLR4) recognition of LPS triggers signalosome assembly among TLR4, sorting (Mal) and signaling (MyD88) adapters, initiating recruitment and activation of kinases, activation of transcription factors, and production of inflammatory mediators. Collaborative studies with investigators at the University of Maryland have shown that tyrosine phosphorylation of Mal regulates its interactions with TLR4, MyD88, interleukin-1 (IL-1) receptor-associated kinase (IRAK)-2 and tumor necrosis factor receptor-associated factor (TRAF)-6 and is important for signaling and is inhibited by endotoxin tolerance. These conclusions were obtained through overexpression of Mal or its variants and mutagenesis of tyrosine residues in HEK293T cells and phosphorylation studies with human monocytes.